High-resolution confocal laser microscopy is an established field in modern imaging and bioimaging technologies. This technique provides sharp, high-magnification, three-dimensional imaging with submicron resolution by non-invasive optical sectioning and rejection of out-of-focus information (see, T. Corle and G. Kino, “Confocal Scanning Optical Microscopy and Related Imaging Systems”, Academic Press, San Diego, 1996). A confocal optical scanner is described in U.S. Pat. No. 5,579,157 to Tanaami et al.
Traditional point scanning confocal systems project a single diffraction limited point of light onto a sample. By imaging that point onto a single element detector, the reflected or fluorescence light originating from that point in the sample can be measured. A single pinhole placed at a conjugate image plane located between the sample and the detector rejects out of focus light and creates the confocal effect. By scanning the point of light in a manner designed to illuminate the focal plane, for example, by raster scanning, an image of the sample can be constructed point by point. By moving the focal plane optically or by moving the sample, multiple focal planes can be imaged and a 3D image constructed.
The use of optical fibers as flexible laser delivery subsystems has been established for many years, proving particularly useful in confocal microscopy. For traditional point scanning confocal microscopy, the only fiber that can be used effectively is a single mode fiber. The light that is emitted from the distal end of a single mode fiber may be considered equivalent to light that is emitted from a diffraction limited source. This fiber tip is re-imaged through the pinhole and onto the sample at or near its diffraction limited size.
A single mode fiber is an optical fiber that is designed for the transmission of a single spatial mode of light as a carrier. This mode of light may contain a variety of different wavelengths, although the range of wavelengths that can be transmitted is a function of the diameter of the core of the fiber. Typical core diameters of single mode fibers are only slightly larger than the wavelengths of light that they transmit. For example, a fiber that transmits in a band around 488 nm is approximately 3.5 microns in core diameter. The cone angle of light that can be coupled into and is emitted from a single mode fiber is characterised by the numerical aperture (NA) of the fiber. The NA of a single mode fiber is a function of the difference between the refractive index of the fiber core and cladding. The distribution of light emitted from a single mode fiber is well approximated by a Gaussian shape, the width of which is determined by the NA of the fiber.
Because of the small diameter of the fiber core, single mode fibers are used most often with laser sources. Other sources of radiation are difficult or impossible to couple into single mode fibers with good efficiency.
A recent development has been the parallel application of the confocal technique. By the use of various optical means, a plurality of near diffraction limited illumination points are projected onto or into the sample. Each of these points is imaged through a corresponding pinhole at a conjugate focal plane onto an image sensor such as a CCD camera. In effect, such a system operates as a plurality of point scanning confocal systems operating in parallel. Several commercial implementations of this concept exist on the market today and can be referred to in general as multiplexed confocal systems.
One implementation of a multiplexed confocal system uses a spinning disk comprising a pattern of several thousand pinholes. An example of one such spinning disk confocal system is one which comprises a Nipkow disk. The use of a multiplexed confocal system employing the Nipkow disk method with microlenses has been disclosed in, for example, U.S. Patent Publication No. 2007/0096014 to Mikuriya et al. The microlenses create a plurality of focal points. A confocal system which creates multiple focal points using microlenses or other focusing means may be referred to as a multi-focal confocal system and forms a subset of multiplexed confocal systems.
In the instrument described in U.S. Patent Publication No. 2007/0096014, the exciting laser light is coupled to the incident end of an optical fiber by a condenser lens and is guided by the optical fiber to an inlet of a confocal scanner unit. A diverging beam of exciting light emitted from the distal end of the optical fiber is converted into a collimated beam by a collimating lens. The collimated beam falls on a disk with a microlens array that focuses excitation laser light onto a pinhole disk (Nipkow disk) mounted on the same axis in such a way that each lens focuses its light onto a corresponding pinhole. Multiple exciting light beams are converged to a sample by an objective lens. Fluorescence and/or reflected light originating from the sample passes through the objective lens again, returns through the same pinholes and is reflected by a dichroic mirror positioned between the microlens disk and the Nipkow disk. The image is then focused onto an image sensor by a relay lens.
In such an apparatus, the Nipkow disk is co-rotated with the microlens disk at a constant speed, and the converged points of light on the sample are scanned with the pinholes moved by the rotation. A plane of the Nipkow disk, a plane to be observed in the sample, and an image sensor plane are arranged to be conjugate with each other optically. Therefore, an optically sectioned image, that is a confocal image of the sample, is formed on the image sensor. Such a system as described above is made by Yokogawa Electric Corporation of Japan and given designations such as CSU-10, CSU-21, CSU-22 and CSU-X1.
Other implementations of multi-focal confocal systems using microlenses exist where the key differences are in the geometry of the microlens patterns and the scanning mechanisms for moving the microlenses and pinholes. An example of such a system is called the Infinity and is built by VisiTech International Ltd. of Sunderland, United Kingdom.
Illumination methods for multi-focal confocal systems are similar to traditional point scanning systems and utilize single mode fibers. In this case, the microlenses image the fiber tip to many parallel pinholes at or near the diffraction limit. The single mode fiber also creates a smooth Gaussian light distribution such that the light distribution between microlenses is relatively uniform. As with confocal point scanning systems, the typical radiation source for multi-focal confocal systems is a laser or multiple lasers coupled through a single mode fiber.
Other means of coupling single mode fibers to multi-focal confocal systems have been proposed.
If only one radiation source is optically coupled to the single mode optical fiber, the radiation source must be changed in order to excite samples using light (from lasers or other radiation sources) with different wavelengths. U.S. Pat. No. 6,603,780 to Miyai illustrates, for example, how laser light is input to a single mode optical fiber by switching between radiation sources with different wavelengths to provide multi-wavelength operation of a confocal microscope. For the above-noted reason, the conventional apparatus has been problematic in that it is not possible to observe different types of fluorescence produced by other types of excitation radiation simultaneously. Another problem is that extra time is required to attach and detach a radiation source to and from the optical fiber. Yet another problem is that vibration arising when the radiation source is attached to or detached from the optical fiber causes the sample to move.
Another approach, disclosed in Japan Patent Publication No. 2003-270543, is to use a plurality of lasers varying in wavelengths coupled to a corresponding plurality of single mode fibers, each distal end of which is provided with an individual collimator coupled to a laser beam-synthesizing mechanism for synthesizing a plurality of the laser beams and making the multi-wavelength laser beam incident as the excitation light on the confocal subsystem. The major disadvantages of this system are the bulkiness and complexity of the beam-synthesizing mechanism, and the very high requirements for thermal and temporal stability of the whole single mode fiber-based light delivery system, its components and subsystems.
Another approach of the prior art, disclosed in U.S. Pat. No. 7,190,514 to Mikuriya et al., is to use a number of lasers coupled to a proportional number of single mode fibers bundled to form a multi-core optical fiber cable. Light that exits the distal end of the multi-core optical fiber cable is collimated by a lens and projected onto a disk of microlenses. The fibers in the bundle being closely spaced (125 microns) provide almost coincident points of light of different wavelengths on the pinhole disk with their misalignment much smaller than a pinhole diameter of 50 microns. As a result, fluorescence observations can be made using a plurality of types of excitation light with the conventional confocal microscope left as is, without the need for attaching and detaching a radiation source to and from the optical fiber.
There are disadvantages to using single mode fibers for some applications. Systems using single mode fibers are, in practice, restricted to radiation sources that emit light with small etendue such as lasers with good beam quality, for example, beam quality factor M2<1.2. Laser sources with good beam quality can be coupled to single mode fibers with coupling efficiencies of approximately 45% to 85% although the efficiency in practice is often less. Lasers with lesser beam quality couple with even lower efficiencies. Single mode fibers can only operate as such over a limited spectral range. Above a given upper cutoff wavelength the fiber is too small to transmit light. Below a lower cutoff wavelength, the light is no longer transmitted in a single mode. The Gaussian distribution of the single mode fiber output intensity is less than ideal for systems requiring even illumination. Only the central part of the Gaussian beam is often used, such that the variation in intensity is less than some amount, for example 20%. In such systems a compromise between evenness in light distribution across an image plane and the light utilization efficiency is required because the peripheral part of the Gaussian beam is abandoned.
Another disadvantage of a system that uses single mode fibers is the requirement for high thermal, mechanical, and temporal stability of the laser-to-fiber alignment and the high manufacturing cost of such a stable system. Designing a means of providing stable laser-to-fiber coupling, and the creation of systems coupling multiple lasers to a single mode fiber, can be challenging.
A different class of multiplexed confocal scanners exists. Multiplexed confocal scanners in that class do not utilize microlenses to focus the light through the corresponding pinholes. Such systems place the Nipkow or similar pinhole disk directly in the collimated light path with no focusing of the light through the pinholes. In some of these systems the pinholes are small slits. These systems are less efficient in their utilization of light as much of the light is blocked by the opaque regions of the Nipkow or similar disk. Such systems do not typically use single mode fibers for coupling light to the microscope and more typically use an arc lamp as the radiation source.
A primary advantage of the multi-focal approach over a multiplexed system as described above is that a greater fraction of the excitation light is directed through the pinholes. This fact provides for greater efficiency but also introduces less scattered light into the optical system which can be a limiting factor in the overall system performance.
“A Mercury Arc Lamp-Based Multi-Color Confocal Real Time Imaging System for Cellular Structure and Function”, Cell Structure and Function, vol. 3, pages 133-141, 2008) by Saito et al. describes the use of a multi-mode fiber with a 1 mm core diameter to couple an arc lamp to a Yokogawa CSU-10. The efficiency of the light coupled from the end of the multi-mode fiber through the CSU is reported to be 1%. It is not clearly defined how this measurement was made but this number represents a low efficiency of light utilization. Saito et al. do not use this fiber with a laser but only with a broadband arc lamp source. Furthermore, with the use of such a large fiber, much of the lost light is scattered from the back surface of the pinhole disk, thus leading to a higher potential for the loss of contrast.